The objective of this research proposal is to elucidate the detailed mechanism of cytochrome c peroxidase catalysis. Emphasis will be given to the electron transfer reactions between the physiological substrate, ferrocytochrome c, and the oxidized enzyme intermediates. Both steady state and transient state kinetic methods will be used to establish the kinetic mechanism. The kinetic studies will be supplemented by investigations of the equilibrium binding between the enzyme and substrate, spectroscopic properties of the enzyme and chemical modifications. The studies are designed to determine whether or not complex formation between substrate and enzyme is required for electron transfer, the possibility of multiple electron transfer pathways and the involvement of specific amino acid residues in the electron transfer pathway.